Anti-Tat MTT Assay: A Novel Anti-HIV Drug Screening System Using the Viral Regulatory Network of Replication

Abstract

Since the recognition of its pivotal role in viral replication, Tat activity has become an interesting target for chemotherapeutic intervention of HIV infection. Here, we report a sensitive and simple colorimetric assay for the screening of Tat inhibitors. We have constructed a plasmid that contains the hygromycin B phosphotransferase gene under the control of the HIV-1 long terminal repeat (LTR) and HIV-1 tat gene constitutively expressed from the cytomegalovirus promoter. This plasmid has been stably transfected to the CD4+ T cell line CEM, which is rendered resistant to hygromycin B through the action of Tat. The inhibitory activity of the anti-Tat drugs was assessed by the extent of cytotoxicity in the presence of hygromycin B as a consequence of the suppressed expression of the hygromycin B phosphotransferase gene. Spectrophotometric quantitation of cell viability was done utilizing 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) dye as the indicator. Using this assay system, we have confirmed that known anti-Tat compound Ro5-3335 and its derivative Ro24-7429 could inhibit Tat-mediated gene expression although their selectivities (anti-Tat activity versus nonselective cytotoxicity) were narrow. Since this method offers the advantage of not handling infectious particles or radioactive materials, it can offer wide applicability as a screening system for anti-Tat compounds.

α‐Lipoic acid blocks HIV‐1 LTR‐dependent expression of hygromycin resistance in THP‐1 stable transformants

“…The findings were analyzed based on the results of a colorimetric cytotoxicity assay using 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) dye. The MTT assay utilized in this investigation was our modification of the original protocol of Mosmann [18] which we designed and established as a screening method for anti-Tat drugs [19]. To further support the findings with the current assay system, we have confirmed the effect of LA and NAC on viral p24 antigen expression in the culture fluid of TNF-c~-stimulated OM 10.1 cells, a macrophage cell line that is latently infected with HIV-1 [20].…”

“…pKO plasmid was used to render the transfected cells resistant to hygromycin B. A detailed description of pKO plasmid construction has been presented previously [19]. However, for this particular experiment, the pKO plasmid was cut by XbaI as to render Tat nonfunctional.…”

“…THP-1 recombinant cells, referred to in this paper as T-Xba, were prepared by transfection of the Xbal-digested pKO plasmid [19]. Electroporation was carried out with Electro Cell Manipulator 600 (BTX, San Diego, CA, USA) under the following conditions: 2950 laF capacitance and 0.5 kV/cm field strength using 2-mm gap BTX cuvette containing 4X 106 cells resuspended in 200/al serum-free RPMI 1640 with 10 mM dextrose and 0.I mM dithiothreitol.…”

“…Thereafter, cells were maintained in RPMI 1640 supplemented with 10% FCS and 300 ~tg/ml hygromycin B (Sigma Chemical Co., St. Louis, MO, USA). As dictated by the restriction site of the transfected plasmid, the expression of the hygromycin phosphotransferase gene is dependent on host transcription factors, particularly NF-~cB [19].…”

“…This has been described in detail in our previous paper [19]. Briefly, 'E' refers to that which causes cytotoxicity due to the effect of compound against NF-~cB, that is, cell death in the presence of both the test drug and hygromycin B.…”